The purification of proteins for the production of biological or pharmaceutical products from various source materials involves a number of procedures. Therapeutic proteins may be obtained from plasma or tissue extracts, for example, or may be produced by cell cultures using eukaryotic or procaryotic cells containing at least one recombinant plasmid encoding the desired protein. The engineered proteins are then either secreted into the surrounding media or into the perinuclear space, or made intracellularly and extracted from the cells. A number of well-known technologies are utilized for purifying desired proteins from their source material. Purification processes include procedures in which the protein of interest is separated from the source materials on the basis of solubility, ionic charge, molecular size, adsorption properties, and specific binding to other molecules. The procedures include gel filtration chromatography, ion-exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography.
Hydrophobic interaction chromatography (HIC) is used to separate proteins on the basis of hydrophobic interactions between the hydrophobic moieties of the protein and insoluble, immobilized hydrophobic groups on the matrix. Generally, the protein preparation in a high salt buffer is loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the proteins in solution, thereby exposing hydrophobic regions in the protein which are then adsorbed by hydrophobic groups on the matrix. The more hydrophobic the molecule, the less salt is needed to promote binding. Usually, a decreasing salt gradient is used to elute proteins from a column. As the ionic strength decreases, the exposure of the hydrophilic regions of the protein increases and proteins elute from the column in order of increasing hydrophobicity. See, for example, Protein Purification, 2d Ed., Springer-Verlag, New York, 176-179 (1988).
When developing processes for commercial production of therapeutically important proteins, increasing the efficiency of any intermediate purification steps is highly desirable. One way of improving the ease and efficiency of manufacturing is to increase the load capacity of one or more of the intermediate steps of the purification process to the point that the number of cycles required to purify a batch of protein is reduced without compromising the quality of the protein separation. The present invention improves the process of protein purification by increasing the capacity and efficiency of an intermediate step.